The overall objective of this proposal is to elucidate the mechanisms whereby hyperglycemia increases myocardial ischemia and reperfusion injury with the goal of reducing cardiovascular morbidity and mortality in patients with diabetes and hyperglycemia. Impaired nitric oxide (NO') signaling is believed to play a key role in the pathogenesis of cardiovascular disease during diabetes and hyperglycemia and endothelial nitric oxide synthase (eNOS) function is a critical factor. Thecurrent proposal will test the overall hypothesis that hyperglycemia impairs cardioprotective signal transduction mechanisms in the heart by attenuating heat shock (HSP)90/eNOS interactions through apathway involving reactive oxygen and nitrogen species and tetrahydrobiopterin (BH4). During Specific Aim 1, we will evaluate the hypotheses that hyperglycemia dose-dependently impairs eNOS coupling and results in eNOS-dependent decreases in NO' and increases in superoxide anion (O2'~) by modulation of HSP90/eNOS chaperone and BH4 co-factor availability in rabbits in vivo and in endothelial cells and cardiomyocytes in vitro. We will address the hypotheses that hyperglycemia-induced formation of peroxynitrite (ONOO") impairs HSP90/eNOS by decreasing phosphotyrosine-HSP90 and increasing nitrotyrosine-HSP90; and by decreasing BH4. Finally, we will evaluate the hypothesis that hyperglycemia blocks ischemic preconditioning (IPC) by attenuating HSP90/eNOS interactions and by decreasing the availability of BH4 in vivo. These experiments will provide novel mechanistic information on the role of HSP90 and hyperglycemia to modulate cardioprotection through protein-protein interactions and altered radical formation using an integrated cellular, molecular and pharmacological approach. During Specific Aim 2, we will address the hypotheses that increasing phosphotyrosine on HSP90 with a phosphotyrosine phosphatase inhibitor; increasing BH4 availability with exogenous BH4 or its metabolic precursor sepiapterin; and decreasing oxidant stress with a novel apo A-1 mimetic D4-F will enhance HSP90 /eNOS association; restore eNOS coupling during hyperglycemia in vitro and in vivo; and restore protection against myocardial infarction produced by IPC in the presence of hyperglycemia through an HSP90-mediated pathway. These experiments will elucidate a novel role for HSP90 during cardioprotection and will confirm potential new therapeutic targets for the treatment of diabetes and hyperglycemia. Lay description: Increases in blood sugar that occur in individuals with diabetes increase the risk of heart attack and death. Theproposed research will evaluate the role of heat shock protein (HSP)90 to promote resistance to heart injury; will determine the adverse effects of blood sugar to impair HSP90 effects: and will identify potential new treatment strategies for diabetes.